An experimental system that enables efficient virus amplification is essential for viral research and research and development of antiviral drugs. Moreover, if a system for amplifying a virus using cultured cells or a system for evaluating viral growth using cultured cells exists, viral research and research and development regarding antiviral drugs will be drastically advanced.
The hepatitis C virus (hereinafter, HCV) belongs to the family Flaviviridae, comprising single-stranded (+) sense RNA as its genome, and is known to cause hepatitis C. Recent studies have revealed that the hepatitis C virus is classified into many types depending on genotype or serotype. According to Simmonds et al's phylogenetic analysis method using the nucleotide sequences of the HCV strains, HCV is classified into 6 genotypes, and the genotypes are further classified into several subtypes (Simmonds, P. et al, Hepatology, 1994, 10: 1321-1324). The nucleotide sequences of the full-length genomes of a plurality of HCV genotypes have now been determined (Choo et al., Science, 1989, 244: 359-362; Kato et al., J. Med. Virol., 2001, 64: 334-339; Okamoto, H et al., J. Gen Virol., 1992, 73: 673-679; and Yoshioka et al., Hepatology, 1992, 16: 293-299).
Until recently, infection of cultured cells with HCV or replication of the HCV genome in cultured cells had been impossible. Hence, research into the HCV replication mechanism or the HCV infection mechanism has required experiments using an in vivo system using chimpanzees as experimental animals. However, preparation of subgenomic replicon RNA from the Con1 strain, the N strain, and the O strain of HCV of genotype 1b, as well as the H77 strain of HCV of genotype 1a has made it possible to conduct experiments on research into the HCV replication mechanism in an in vitro system using cultured cells (JP Patent Publication (Kokai) No. 2001-17187 A; Lomann et al., Science, 1999, 285: 110-113; Blight et al., Science, 2000, 290: 1972-1974; Friebe et al., 2001, 75: 12047-12057; and Ikeda et al., J. Virol., 2002, 76: 2997-3006). Here, the term “HCV subgenomic replicon RNA” refers to RNA comprising a part of the HCV genome, which is incapable of producing infectious HCV particles but capable of self-replication of HCV genome-derived RNA introduced into cells.
Furthermore, together with subgenomic replicon RNA, full-genomic replicon RNA, by which infectious HCV particles are produced by in vitro introduction into Huh7 cells, has been prepared from the JFH-1 strain of HCV of genotype 2a. This has made it possible to conduct an experiment with an in vitro system using cultured cells also for research into the HCV infection mechanism (Kato, T et al., Gastroenterology, 2003, 125: 1808-1817; and Wakita, T et al., Nat. Med., 2005, 11: 791-796). Here, the term “HCV full-genomic replicon RNA” refers to RNA comprising a full-length HCV genome, which is capable of self-replication of HCV genome-derived RNA introduced into cells and is capable of producing infectious HCV particles.
Meanwhile, hepatitis C is currently treated mainly by single-agent therapy with interferon-α or interferon-β and combination therapy with interferon-α and ribavirin, which is a purine-nucleoside derivative. However, it is known that even when these therapies are carried out, therapeutic effects are observed for only about 60% of all treated patients. It is also known that the disease flares up again among half or more of effectively treated patients if the therapies are discontinued. Also, the therapeutic effects of interferons are associated with HCV genotypes and thus are known to be low for genotype 1b but high for genotype 2a (Mori, S., et al., Biochem. Biophis. Res. Commun., 1992, 183: 334-342).
The reasons why the therapeutic effects of interferons differ depending on HCV genotype have not yet been clarified. One of the reasons is thought to be the presence of differences in HCV replication mechanism or HCV replication efficiency.
However, the presence of the HCV subgenomic replicon RNA is limited to several types from the HCV strains of genotypes 1a, 1b, and 2a. Moreover, the presence of full-genomic replicon RNA is limited to one type from the JFH-1 strain of HCV of genotype 2a. Hence, elucidation of the relationship between HCV genotype and HCV replication mechanism or HCV replication efficiency has been difficult. Also, the types of viral particles that can be artificially prepared and used for raw materials of HCV vaccines are also limited to those generated from the full-genomic replicon RNA. Thus, the finding of other subgenomic replicon RNAs or full-genomic replicons RNA of HCV with a characteristic replication mechanism or replication efficiency has been desired.
Subgenomic replicon RNAs or full-genomic replicon RNAs from HCV of the same genotype or from the same HCV strain having different characteristics in terms of replication mechanism or replication efficiency have been absent. Hence, differences in HCV replication mechanism or HCV replication efficiency could not have been compared using samples with the same genetic background. Furthermore, factors required for replication of HCV targeted by a new anti-HCV therapeutic agent could not have been identified and an anti-HCV therapeutic agent capable of exerting beneficial effects independently from the replication mechanism or the replication efficiency could not have been screened for.